Interpretive Summary: The identification of proteins involved in the insect (aphid) transmission of Barley Yellow Dwarf Virus (BYDV) is critical to the development of novel virus disease control strategies. The current research was initiated to investigate protocols for the isolation of aphid proteins. The most useful protocols will allow the extraction and separation of a wide diversity of proteins that are reproducible among repeated experiments. To examine protein diversity, approximately 600 proteins from aphids were identified using mass spectrometry, a technique that accurately measures the mass of individual proteins. To examine reproducibility, numerous characteristics of each of three protein extraction methods were compared including, protein concentration, protein diversity and technical variability. Only one of the three methods provided both significant protein diversity and quantitative reliability, but each method also identified unique proteins. Therefore the results demonstrate the importance of using multiple protein extraction methods to identify the widest spectrum of proteins. Also, by optimizing the protein extraction methods more reliable data was generated that will facilitate the continuation of studies to identify insect proteins responsible for the transmission of viruses that have a high agricultural and economic impact.

Technical Abstract:
Few attempts have been made to methodically compare protein isolation methods from insect tissues for proteomic studies. To address this, we compared qualitative and quantitative differences among three methods for isolation, purification and solubilization of insect proteins. Schizaphis graminum, an aphid pest was the source of insect tissue. Proteins were extracted using either trichloroacetic acid in acetone (TCA-A), a phenol extraction, or a multidetergent extraction, solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis. The TCA-A method had the highest protein yield as well as the greatest number of total proteins from aphid tissues. There were numerous differences in the 2-D gel spot pattern between the different extractions. Mass spectrometry was used to identify proteins from each extraction type; however, there were no functional or biochemical explanations for the differences in protein extraction from each extraction type. The TCA-A method was further explored for its quantitative reliability using DIGE and a principal component analysis showed little of the variation in the data was due to technical issues, thus demonstrating it is an ideal method for preparing proteins for a quantitative proteomics experiment. These data suggest that, while the TCA-A method outperformed the phenol and the multigetergent methods for extracting aphid proteins, a combination of extraction approaches would be ideal for increasing proteome coverage when using gel-based separation techniques.